1. Field of the Invention
The present invention relates to the use as therapeutic agents of IL-2, IL-2 peptides and derivatives which act as interleukin-2 (IL-2) agonists and antagonists, and to methods of modulating IL-2 activity.
2. Discussion of the Background
Interleukin-2 (IL-2) is the main growth factor of T lymphocytes (Thxc3xa8ze et al. 1996, Immunol. Today 17:481-486). By regulating T helper lymphocyte activity IL-2 increases the humoral and cellular immune responses. By stimulating cytotoxic CD8 T cells and NK cells this cytokine participates to the defense mechanisms against tumors and viral infections. IL-2 is used in therapy against metastatic melanoma and renal adenocarcinoma. IL-2 is used in clinical trials in many forms of cancer (Lotze and Rosenberg 1988, Interleukin 2 as a Pharmaclologic Reagent. in Interleukin 2, K. A. Smith, Academic Press: p. 237-89). It is also used in HIV infected patients and leads to a significant increase in CD4 counts (Kovacs et al. 1996, New Engl. J. of Medicine 1350-6).
Human IL-2 is a protein of 133 amino acids (aa) composed of four xcex1 helices connected by loops of various length; its tridimensional structure has been established. IL-2R is composed of three chains xcex1, xcex2 and xcex3. IL-2Rxcex1 controls the affinity of the receptor. IL-2Rxcex1 and IL-2Rxcex3 are responsible for IL-2 signal transduction. The different molecular areas of IL-2 interacting with the three chains of the IL-2 R have been defined. More specifically it has been determined that a helix A as well as the NH2 terminal area of IL-2 (residues 1 to 30) control the interactions IL-2/IL-2Rxcex2 (Eckenberg et al. 1997, Cytokine 9:488-98): IL-2Rxcex2 chain is the most important in IL-2 signaling (Thxc3xa8ze et al. 1990).
The effects of human interleukin-2 (IL-2) on its target cells are mediated through specific cell surface receptors (IL-2R) (Taniguchi et al. (1983) Nature 302:305-310; Robb et al. (1984) Proc. Natl. Acad. Sci. USA 81:6486-6490; Smith K A. 1988a. Interleukin-2; Smith K A (1988b) Science 240:1169-1176). IL-2R comprises at least three subunits encoded by different genes (Minami et al. (1993) Annu. Rev. Immunol. 11:245-267; Taniguchi et al. (1993) Cell 73:5-8). The first component to be identified, IL-2Rxcex1, is a 55 kDa protein that binds IL-2 with a Kd of ≅10 nM (Uchiyama et al. (1981) J. Immunol. 126:1293-1297; Leonard et al. (1984) Nature 311:626-631). The role of IL-2Rxcex1 (Kumar et al. (1987) J. Immunol. 139:3680-3684) and the influence of IL-2 on IL-2Rxcex1 gene expression have been studied (Bismuth et al. (1985) Eur. J. Immunol. 15:723-727; Froussard et al. (1991) Mol. Immunol. 28:87-93). The second IL-2R component, IL-2Rxcex2 is a 75 kDa protein with a large intracytoplasmic domain (286 aa) (Teshigawara et al. (1987) J. Exp. Med. 165:223-238; Hatakeyama et al. (1989) Science 244:551-556; Tsudo et al. (1989) Proc. Natl. Acad. Sci. USA 86:1982-1986). The last component to be identified, IL-2Rxcex3, is a 64 kDa protein (Takeshita et al. (1992) Science 257:379-382; Ishii et al. (1994) Int. Immunol. 6:1273-1277). IL-2Rxcex2 and IL-2Rxcex3 belong to the hematopoietin receptor family whereas IL-2Rxcex1 belongs to another family of molecules (Thxc3xa8ze J (1994) Eur. Cytokine Netw. 5:353-368). In the mouse system all three chains are required to form a functional receptor (Moreau et al. (1995a) J. Immunol. 155:3401-3408; Chastagner et al. (1996) Eur. J. Immunol. 26:201-206). In the human system two receptors are functional. When associated, human IL-2Rxcex2 plus IL-2Rxcex3 form an intermediate affinity receptor with a Kd of ≅1 nM, whereas expression of all three chains leads to the formation of a high affinity IL-2R (Kd≅10 pM).
The structure of IL-2 (MacKay D (1992) Science 257:410-413) is composed of a compact core bundle of four antiparallel xcex1 helices connected by three loops (FIG. 1). Some of the interactions between IL-2 and IL-2Rxcex1 (Sauv et al. (1991) Proc. Natl. Acad. Sci. USA 88:4636-4640; Wang et al. (1995) Eur. J. Immunol. 25:1212-1216) and IL-2Rxcex3 subunits (Voss et al. (1993) Proc. Natl. Acad. Sci. USA 90:2428-2432; Buchli et al. (1993) Arch. Biochm. Biophys. 307:411-415) have been elucidated, but less is known concerning IL-2/IL-2Rxcex2 interaction, despite the fact that IL-2Rxcex2 chain plays a very critical role in signal transduction (Taniguchi T (1995) Science 268:251-255).
It has been shown that one substitution Asp20 by Lys (mutant D20K) prevents binding to IL-2Rxcex2 (Collins et al. (1988) Proc. Natl. Acad. Sci. USA 85:7709-7713). In a recent report the role of the sequence (Leu17, Leu18, Leu19, Asp20, Leu21) from IL-2xcex1 helix A, in IL-2/IL-2Rxcex2 interactions was analyzed by cassette mutagenesis (Berndt et al. (1994) Biochemistry 33:6571-6577). However the data were difficult to interpret since most of the proteins produced have multiple mutations inside and outside of the sequence of interest. Only one analog with a single mutation was studied (L21V). More surprisingly it was reported in this study that deletion of the segment spanning residues 17-31 (Del1) gives a protein with full agonist activity.
IL-2 peptides and derivatives were described in Cytokine (1997) 7:488-498, but were not tested in an in vitro system for biochemical activity such as cytokine activity, and in particular for IL-2-like activity.
In view of the aforementioned deficiencies attendant with the prior art analysis of IL-2 agonists and antagonists, as well as with methods of modulating IL-2 activity therewith, it is clear that there exists a need in the art for the same.
Accordingly, one object of this inventions to provide compositions having an IL-2-like activity and methods for their use as therapeutic agents. The applications of such recombinant, synthetic or hybrid peptides are thus one object of the invention. These compositions are defined as having the following characteristics: a) containing one or more peptides at least five amino acids in length; and b) inhibiting or mimicing the binding of xcex1 helix A of interleukin-2 (IL-2) to a xcex2 subunit of an IL-2 receptor (IL-2R).
Another object of the invention is the use of a purified peptide having the following characteristics: a) the peptide is at least five amino acids in length; b) the peptide binds to a xcex2 subunit of an IL-2 receptor (IL-2R); and c) the peptide induces phosphorylation of the xcex2 subunit of the IL-2R.
A further object of the invention concerns the preparation of the antibodies which recognize the peptides of the invention, and the therapeutic use of these antibodies.
A further object of the invention is the use of DNA sequences encoding the peptides of the invention and their derivatives. Such DNA fragments are useful for gene therapy among other applications. The use of a DNA of the following sequence
ATG GCT CCG ACG AGC AGC TCC ACC AAG AAA ACC CAG CTC CAG CTC GAA
CAC CTG CTG CTG GAC CTG CAG ATG ATC CTG AAC GGT ATC AAC AAC
(SEQ ID NO:1) is one particular object of the invention.
Yet another object of the invention is to provide a method for detecting the activity of an IL-2-like peptide, wherein the IL-2 activity is measured by the binding of the IL-2R to the peptide having the IL-2 agonist or antagonist activity.
A still further object of the invention is the use of compounds which inhibit the activity of an IL-2R by contacting the IL-2R with an amount of the selected antagonist peptide sufficient to inhibit binding of IL-2 to the IL-2R under conditions that allow binding of the peptide to the IL-2R to occur.
Another object of the invention is to provide a method for the selection of antibodies specific for the purified peptide with IL-2-like activity as defined herein. These monoclonal or polyclonal antibodies can inhibit binding of IL-2 to the IL-2R under conditions that allow binding of the peptide to the IL-2R to occur. The therapeutic use of these antibodies is also a part of the present invention. In particular, these antibodies specific for the purified peptides are useful for treating or preventing undesirable immune reactions such as graft rejection or autoimmune disorders, for example, rheumatoid arthritis.
A still further object of the invention is to provide a method for inducing in a patient the biological effects of IL-2 by administering to the patient an amount of the agonist peptide of the invention sufficient to induce those biological effects, or by administering a combination of various cytokines and purified peptide. By various cytokines is meant for example IL-4, IL-9, IL-15 or IL-2.
Another object of the invention relates to the nucleic acid sequences corresponding to the amino acid sequence of the purified peptide and its derivatives according to the invention. A preferred embodiment is the nucleotide sequence encoding the purified peptide IP130 having the following sequence (SED ID NO:2):
Met Ala Pro Tbr Ser Ser Ser Thr Lys Lys Thr Gln eu Gln Leu Glu
His Leu Leu Lea Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn (SEQ ID NO: 2) This sequence or a sequence derived therefrom can be inserted in an appropriate vector capable of expressing the product in vivo, in a bacterium or in a eukaryotic cell, particularly in yeast or a mammalian cell. These constructs are useful for gene therapy among other uses.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.